Project Summary/Abstract Obesity as a major public health issue has increased the interest in low-calorie, natural sweeteners. A prime candidate is the 53-residue, heat stable, protein, brazzein, which contains no carbohydrate and is perceived as sweet tasting only by old world primates and humans. Through mutagenesis we have determined that three major regions near N- and C-terminal domains and Loop43 region are critical for heterodimeric sweet receptor binding and/or activity; mutations in other regions appear to affect sweetness by indirect conformational changes, as detected by NMR spectroscopy. Complementary mutagenesis studies of the sweet receptor indicate that brazzein interacts with specific residues in the cysteine-rich domain (CRD) of the T1R3 subunit. In addition, modeling and receptor mutagenesis studies have identified a major binding surface for brazzein on the VFTM (Venus flytrap ligand binding extracellular module) of T1R2 subunit. This large interaction surface on the receptor distinguishes the mode of action of brazzein from those of small molecule sweeteners. Specific contributions of T1R2 residues in determining the differential sensitivity of the receptor to brazzein remain to be discovered. We propose three specific aims: 1. In vitro cellular assays of sweet protein-receptor interactions. 2. To use NMR spectroscopy to investigate the conformational and dynamic requirements in brazzein for its interaction and activation of the sweet taste receptor. These studies will serve to identify changes that correlate with functional properties. 3. To monitor binding of brazzein and its mutants to T1R2/T1R3 sweet receptor and its mutants by STDD-NMR spectroscopy. These results will contribute to accurately define the essential molecular features responsible for the brazzein- sweet receptor interaction and the resulting signal transduction non-caloric sweeteners as an approach to help addressing diabetes and related disorders. PUBLIC HEALTH RELEVANCE Obesity as a major public health issue has increased the interest in low-calorie, natural sweeteners. A prime candidate is the 53-residue, heat stable, protein, brazzein, which contains no carbohydrate and is perceived as sweet tasting only by old world primates and humans. Explanation of molecular features that are essential for the brazzein-sweet receptor interaction may result in design of superior tasting non-caloric sweetener as an approach to help addressing diabetes and related disorders.